In ovo vaccination against coccidiosis

ABSTRACT

The invention relates to a method of vaccinating a domesticated bird against coccidiosis comprising administering in ovo an effective immunizing dose of live Eimeria sporocysts or oocysts, or a mixture thereof.

This application is a 371 of PCT/IB95/00445 filed Jun. 7, 1995.

BACKGROUND OF THE INVENTION

The present invention relates to a method of vaccinating domesticatedbirds against coccidiosis. In particular, the invention relates to thein ovo administration of live Eimeria spp sporocysts or oocysts, ormixtures thereof, into the developing eggs of domesticated birds inorder to immunize the hatched chicks against coccidiosis.

Coccidiosis is an enteric disease of domesticated birds caused byinfection with intracellular protozoan parasites of the genus Eimeria.Coccidiosis is the most economically devastating parasitic disease ofdomesticated birds. It is estimated that anticoccidial medications andlosses due to coccidiosis cost the poultry industry hundreds of millionsof dollars every year.

Various attempts to vaccinate domesticated birds against coccidiosishave been reported since the early 1950's. Current vaccination methodsinclude administering live Eimeria oocysts to birds through feed orwater. These methods, however, are inconvenient and inefficient becausenot all birds get the intended oocyst dose and many are eitherunprotected by the vaccine or receive a pathogenic infection.

In J. M. Sharma and B. R. Burmester, Avian Dis. 26: 134-149, 1981, theauthors reported that chickens vaccinated in ovo with herpesvirus ofturkey developed immunity against subsequent challenge with Marek'sdisease virus. In European patent publication no. 291173, animmunization process is referred to wherein a nonreplicating immunogenis administered in ovo. The immunogens specifically referred to in theEuropean patent are a genetically engineered Eimeria antigen and anEimeria oocyst extract. The European patent specifically excludes liveparasite stages such as those used in the vaccination method claimedherein.

The present vaccination method involves in ovo administration of liveEimeria sporocysts or oocysts, or a mixture thereof, into the developingeggs of domesticated birds. The available literature suggests that sucha vaccination method would be ineffective in ovo and should be appliedpost-hatch. In T. K. Jeffers and G. E. Wagenbach, J. Parasit. 56(4):656-662, 1970, the authors reported that in ovo injection of E. tenellasporozoites on day 10 of incubation provided no significantimmunological protection against subsequent challenge with E. tenellaoocysts. In fact, they reported that chicks that received no treatmenthad a greater survival rate against subsequent challenge with E. tenellaoocysts than chicks that had been treated in ovo with sporozoites. In K.L. Watkins et al., Proc. VI th. International Coccidiosis Conf.,Abstract E1-2, Ontario, Canada, 1993, the authors described in ovoinoculation with live E. maxima sporocysts and sporulated oocysts,although they did not indicate when the inoculation was done duringincubation of the egg. Watkins et al. concluded that their studyprovided no evidence that in ovo exposure protects against subsequentcoccidial challenge with E. maxima oocysts 10 days post-hatch. They alsoconcluded that significant immunological protection is provided ifinoculation is done soon after hatch rather than in ovo. Contrary tothis teaching, the in ovo vaccination method of the present inventionprovides unexpected immunity that protects the hatched birds againstsubsequent coccidial challenge.

SUMMARY OF THE INVENTION

The present invention, also referred to herein as the “presentvaccination method”, relates to a method of vaccinating a domesticatedbird against coccidiosis comprising administering in ovo, during thefinal quarter of incubation, an effective immunizing dose of liveEimeria sporocysts or oocysts, or a mixture thereof.

The term “domesticated bird(s)”, as used herein, unless otherwiseindicated, includes chickens, turkeys, ducks, game birds (including, butnot limited to, quail, pheasants, and geese) and ratites (including, butnot limited to, ostrich).

The term “in ovo”, as used herein, unless otherwise indicated, meansinto a domesticated bird egg containing a live, developing embryo.

The term “administering in ovo” or “in ovo administration”, as usedherein, unless otherwise indicated, means administering the vaccinedescribed herein to a domesticated bird egg containing a live,developing embryo by any means of penetrating the shell of the egg andintroducing the vaccine. Such means of administration include, but arenot limited to, injection of the vaccine.

The term “final quarter of incubation”, as used herein, unless otherwiseindicated, means the final quarter of incubation of a developing egg ofa domesticated bird.

The term “Eimeria”, as used herein, unless otherwise indicated, meansone or more species of the genus Eimeria that infect domesticated birds.Such Eimeria species include those that are found in chicken, includingE. tenella, E. acervulina, E maxima, E. necatrix, E. mitis, E. praecox,and E. brunetti, and also those that are found in turkeys, including E.meleagrimitis, E. adenoeides, E. gallopavonis, E. dispersa, E.meleagridis, E. innocua, and E. subrotunda, and also Eimeria speciesthat infect other domesticated birds as defined above. The term“Eimeria” also includes all strains of the foregoing species of Eimeria,including, but not limited to, precocious strains, and attenuatedstrains, which includes strains that have been irradiated, or otherwisetreated, so that they fail to complete development. The term Eimeriaalso includes any newly-discovered strains or species of Eimeria thatinfect domesticated birds as defined above.

The term “sporocysts”, as used herein, unless otherwise indicated, meanslive Eimeria sporocysts.

The term “oocysts”, as used herein, unless otherwise indicated, meanslive Eimeria sporulated oocysts or a mixture of sporulated andunsporulated oocysts.

The term “effective immunizing dose”, as used herein, unless otherwiseindicated, means a number of sporocysts or oocysts, or, when mixed, anumber of sporocysts and oocysts, sufficient to provide immunologicalprotection in the hatched birds that is greater than the inherentimmunity of non-immunized birds. As used herein, the terms “immunize”and “vaccinate” are synonymous and are used interchangeably.

A preferred dose to be administered in accord with the method of theinvention comprises 10² to 10⁸ sporocysts or oocysts, or a mixturethereof wherein the total number of said sporocysts and oocysts rangesfrom 10² to 10⁸.

A more preferred dose comprises 10² to 10⁷ sporocysts or oocysts, or amixture thereof wherein the total number of said sporocysts and oocystsranges from 10² to 10⁵.

Another preferred dose comprises 10⁵ to 10⁷ sporocysts or oocysts, or amixture thereof wherein the total number of said sporocysts and oocystsranges from 10⁵ to 10⁷.

Another preferred dose comprises 10⁴ to 10⁶ sporocysts or oocysts, or amixture thereof wherein the total number of said sporocysts and oocystsranges from 10⁴ to 10⁶.

Another preferred dose comprises 10³ to 10⁶ sporocysts or oocysts, or amixture thereof wherein the total number of said sporocysts and oocystsranges from 10³ to 10⁶.

A preferred domesticated bird to be vaccinated in accord with the methodof the invention is a chicken.

A preferred dose to be administered in ovo to chicken eggs comprisessporocysts or oocysts, or a mixture thereof, of two or more species ofEimeria selected from the group consisting of E. tenella, E. acervulina,E. maxima, E. necatrix, E. mitis, E. praecox, and E. brunetti.

Another preferred domesticated bird to be vaccinated in accord with themethod of the invention is a turkey.

A preferred dose to be administered in ovo to turkey eggs comprisessporocysts or oocysts, or a mixture thereof, of two or more species ofEimeria selected from the group consisting of E. meleagrimitis, E.adenoeides, E. gallopavonis, E. dispersa, E. meleagridis, E. innocua,and E. subrotunda.

Other preferred domesticated birds to be vaccinated in accord with themethod of the invention are game birds, ducks and ratites.

The method of the invention further comprises, in combination withpresent vaccination method, administering in ovo an immune stimulant atany time during incubation.

A preferred method of administering the immune stimulant issimultaneously with the in ovo administration of a dose of sporocysts oroocysts, or mixture of said sporocysts and oocysts, during the finalquarter of incubation.

DETAILED DESCRIPTION OF THE INVENTION

The present vaccination method involves the in ovo administration,during the final quarter of incubation, of live Eimeria sporocysts oroocysts, or a mixture thereof, into domesticated birds' eggs. In thecase of chickens, in ovo administration is preferably done on days 15-20of incubation, and most preferably on day 18 of incubation. In the caseof turkeys, In ovo administration Is preferably done on days 21-26 ofincubation.

The present vaccination method can be performed using any suitable inovo administration method. Preferably, the present vaccine Isadministered via injection. According to one method of injection, a holeis made in the egg shell at the large end of the egg using an 18 guageneedle to expose the egg's air cell. A 1.0-1.5 inch 22 guage needleattached to a syringe of appropriate size (1-3 ml) can be insertedthrough the hole and through the membrane of the air cell. Anappropriate number of sporocysts or oocysts, or, when mixed, anappropriate number of sporocysts and oocysts, are suspended in asuitable liquid carrier, for instance 10-500 μl of phosphate-bufferedsaline, and then injected into the egg. The appropriate volume willdepend on the size of the egg being treated, with ostrich eggs obviouslybeing capable of taking more volume than chicken eggs. The site ofinjection can be within any region of the egg. Preferably, injection isdone axially through the center of the large end of the egg into theamnion.

Alternatively, an automated egg injection system can be used in thepresent vaccination method. Such systems are described in U.S. Pat. Nos.4,681,063, 4,040,388, 4,469,047, and 4,593,646, which are hereinincorporated by reference. Other appropriate methods of injection areknown to those skilled in the art.

Oocysts to be used in accord with the present vaccination method can beprepared by any of several methods known to those skilled in the art.Such methods include those described in J. F. Ryley et al., Parasitology73:311-326, 1976 and P. L. Long et al., Folia Veterinaria Latina VI#3,201-217, 1976, which are herein incorporated by reference. According toone method, commercial broiler chickens, approximately 2 weeks old, areinfected with the Eimera species of interest by oral gavage of anappropriate dose of sporulated oocysts. For example, a typical dose usedfor E. tenella is 200,000 sporulated oocysts/bird. Well known proceduresfor collection and purification of oocysts from infected birds are thenfollowed. For most species of Eimeria, feces are collected from infectedbirds 5-7 days post-infection, blended and filtered to remove debris,then centrifuged at a speed sufficient to pellet the remaining fecalmaterial. For E. tenella, a similar procedure is used except that cecalcores are taken at 6 days post-infection. The pellet is resuspended in asaturated salt solution, in which the oocysts float and most of thecontaminating debris can be removed by centrifugation. The oocystsuspension is then diluted to lower the salt concentration. The oocystsare washed repeatedly to remove the salt and resuspended in potassiumdichromate solution (2.5% w/v). The oocyst suspension is incubated at29° C. with shaking (e.g., 140 rpm) for approximately 72 hours to inducesporulation of the oocysts. Alternatively, the oocysts can be treatedwith sodium hypochlorite and then sporulated. The number of sporulatedoocysts/ml is determined by direct count using a hemocytometer, and theculture is stored, preferably under refrigeration until needed. Theoocysts can be used in accord with the present vaccination method,preferably in a dose comprising from 10² to 10⁸ oocysts per egg. Morepreferably, the dose comprises from 10² to 10⁵ oocysts per egg.

To prepare sporocysts, the potassium dichromate is removed from theoocyst suspension described above by repeated washing of the. oocysts,which involves collection of oocysts by centrifugation and resuspendingin deionized or distilled water. When the dichromate has been removed asjudged by the lack of yellowish-orange coloration, the oocyst suspensionis mixed with an equal volume of sodium hypochlorite (bleach) andincubated at room temperature for 15 minutes. The bleach is then removedby repeated washings, and the oocysts are resuspended in physiologicalsaline or deionized water. Oocysts can be broken to release sporocystsusing a variety of known techniques. For example, oocysts can be brokento release sporocysts by mixing the oocysts with glass beads of 1-4 mmdiameter and shaking by hand, vortex mixer, or shaking incubator, orusing a hand-held homogenizer. Unbroken oocysts and oocysts walls can beseparated from the released sporocysts by differential centrifugation in50% PERCOLL, a colloidal suspension of polyvinyl pyrrolidone coatedsilica particles (sold by Pharmacia Biotech) or 1 M sucrose as describedin Dulski et al., Avian Diseases, 32: 235-239, 1988. The sporocysts canbe used in the present vaccination method either mixed with or separatedfrom the unbroken oocysts and oocysts walls. Preferably, the dose ofsporocysts is separated from the oocysts and oocysts walls. A preferreddose of sporocysts comprises from 10² to 10⁸ sporocysts per egg. Morepreferably, the dose comprises from 10² to 10⁵ sporocysts per egg.

The sporocysts or oocysts, or mixture thereof, can be injected in ovo inany physiologically suitable medium. Preferably, they are suspended inphysiologically balanced saline such as phosphate buffered saline. Theselected medium can optionally include one or more suspending agentsincluding physiologically suitable gels, gelatins, hydrosols, cellulose,or polysaccharide gums.

Preferably, in the present vaccination method, sporocysts or oocysts, ora mixture thereof, of two or more Eimeria species are injected in ovo atthe same time. In accord with the present vaccination method, sporocystsor oocysts, or a mixture thereof, of all identified species of Eimeriathat infect a specific domesticated bird, such as chicken, can beinjected in ovo at the same time, or in series, at appropriate doses toprovide immunological protection against all species.

Immune stimulants can be used in conjunction with the presentvaccination method. Immune stimulants that can be used in the presentvaccination method include, but are not limited to, cytokines, growthfactors, chemokines, supernatants from cell cultures of lymphocytes,monocytes, or cells from lymphoid organs, cell preparations or cellextracts (e.g. fixed Staphylococcus aureus or lipopolysaccharidepreparations), mitogens, or adjuvants, including low molecular weightpharmaceuticals. Immune stimulants can be administered in ovo at anytime during incubation. Preferably, immune stimulants are administeredin ovo in the medium containing the dose of Eimeria sporocysts oroocysts, or mixture thereof.

We claim:
 1. A method of vaccinating a domesticated bird againstcoccidiosis comprising administering in ovo, during the final quarter ofincubation, an effective immunizing dose of live Eimeria sporocysts oroocysts, or a mixture thereof.
 2. The method of claim 1 wherein the dosecomprises 10² to 10⁸ sporocysts or oocysts, or a mixture thereof whereinthe total number of said sporocysts and oocysts ranges from 10² to 10⁸.3. The method of claim 1 wherein the dose comprises 10² to 10⁵sporocysts or oocysts, or a mixture thereof wherein the total number ofsaid sporocysts and oocysts ranges from 10² to 10⁵.
 4. The method ofclaim 1 wherein the dose comprises 10⁵ to 10⁷ sporocysts or oocysts, ora mixture thereof wherein the total number of said sporocysts andoocysts ranges from 10⁵ to 10⁷.
 5. The method of claim 1 wherein thedose comprises 10⁴ to 10⁶ sporocysts or oocysts, or a mixture thereofwherein the total number of said sporocysts and oocysts ranges from 10⁴to 10⁶.
 6. The method of claim 1 wherein the dose comprises 10³ to 10⁶sporocysts or oocysts, or a mixture thereof wherein the total number ofsaid sporocysts and oocysts ranges from 10³ to 10⁶.
 7. The method ofclaim 2 wherein the domesticated bird is a chicken.
 8. The method ofclaim 7 wherein the dose comprises sporocysts or oocysts, or a mixturethereof, of two or more species of Eimeria selected from the groupconsisting of E. tenella, E. acervulina, E. maxima, E. necatrix, E.mitis, E. praecox, and E. brunette.
 9. The method of claim 8 wherein thedose is administered by in ovo injection.
 10. The method of claim 2 andfurther comprising administering in ovo an immune stimulant at any timeduring incubation.
 11. The method of claim 10 wherein the immunestimulant is administered in ovo simultaneously with the dose ofsporocysts or oocysts, or mixture of said sporocysts and oocysts. 12.The method of claim 2 wherein the dose comprises oocysts.
 13. The methodof claim 2 wherein the dose comprises sporocysts.
 14. The method ofclaim 13 wherein the sporocysts have been purified to remove oocysts.15. The method of claim 2 wherein the domesticated bird is a turkey. 16.The method of claim 15 wherein the dose comprises sporocysts or oocysts,or a mixture thereof, of two or more species of Eimeria selected fromthe group consisting of E. meleagrimitis, E. adenoeides, E.gallopavonis, E. dispersa, E. meleagridis, E. innocua, and E.subrotunda.
 17. The method of claim 16 wherein the dose is administeredby in ovo injection.
 18. The method of claim 2 wherein the domesticatedbird is selected from the group consisting of a game bird, duck andratite.